Methods of therapeutic monitoring of nitrogen scavenging drugs

ABSTRACT

The present disclosure provides methods for evaluating daily ammonia exposure based on a single fasting ammonia blood level measurement, as well as methods that utilize this technique to adjust the dosage of a nitrogen scavenging drug, determine whether to administer a nitrogen scavenging drug, and treat nitrogen retention disorders.

RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 13/417,137, filed Mar. 9, 2012 and now pending, which claims thebenefit of U.S. Provisional Application No. 61/564,668, filed Nov. 29,2011, and U.S. Provisional Application No. 61/542,100, filed Sep. 30,2011, the disclosures of which are incorporated by reference herein intheir entirety, including drawings.

BACKGROUND

Nitrogen retention disorders associated with elevated ammonia levelsinclude urea cycle disorders (UCDs) and hepatic encephalopathy (HE).

UCDs include several inherited deficiencies of enzymes or transportersnecessary for the synthesis of urea from ammonia, including enzymesinvolved in the urea cycle. The urea cycle is depicted in FIG. 1, whichalso illustrates how certain ammonia-scavenging drugs act to assist inelimination of excessive ammonia. With reference to FIG. 1, N-acetylglutamine synthetase (NAGS)-derived N-acetylglutamate binds to carbamylphosphate synthetase (CPS), which activates CPS and results in theconversion of ammonia and bicarbonate to carbamyl phosphate. In turn,carbamyl phosphate reacts with ornithine to produce citrulline in areaction mediated by ornithine transcarbamylase (OTC). A second moleculeof waste nitrogen is incorporated into the urea cycle in the nextreaction, mediated by arginosuccinate synthetase (ASS), in whichcitrulline is condensed with aspartic acid to form argininosuccinicacid. Argininosuccinic acid is cleaved by argininosuccinic lyase (ASL)to produce arginine and fumarate. In the final reaction of the ureacycle, arginase (ARG) cleaves arginine to produce ornithine and urea. Ofthe two atoms of nitrogen incorporated into urea, one originates fromfree ammonia (NH₄ ⁺) and the other from aspartate. UCD individuals bornwith no meaningful residual urea synthetic capacity typically present inthe first few days of life (neonatal presentation). Individuals withresidual function typically present later in childhood or even inadulthood, and symptoms may be precipitated by increased dietary proteinor physiological stress (e.g., intercurrent illness).

Hepatic encephalopathy (HE) refers to a spectrum of neurologic signs andsymptoms believed to result from hyperammonemia, which frequently occurin subjects with cirrhosis or certain other types of liver disease.Subjects with HE typically show altered mental status ranging fromsubtle changes to coma, features similar to subjects with UCDs.

Subjects with nitrogen retention disorders whose ammonia levels and/orsymptoms are not adequately controlled by dietary restriction of proteinand/or dietary supplements are generally treated with nitrogenscavenging agents such as sodium phenylbutyrate (NaPBA, approved in theUnited States as BUPHENYL® and in Europe as AMMONAPS®) or sodiumbenzoate. These are often referred to as alternate pathway drugs becausethey provide the body with an alternate pathway to urea for excretion ofwaste nitrogen (Brusilow 1980; Brusilow 1991). NaPBA is a phenylaceticacid (PAA) prodrug. Another nitrogen scavenging drug currently indevelopment for the treatment of nitrogen retention disorders isglyceryl tri-[4-phenylbutyrate](HPN-100), which is described in U.S.Pat. No. 5,968,979. HPN-100, which is commonly referred to as GT4P orglycerol PBA, is a prodrug of PBA and a pre-prodrug of PAA.

HPN-100 and NaPBA share the same general mechanism of action: PBA isconverted to PAA via beta oxidation, and PAA is conjugated enzymaticallywith glutamine to form phenylacetylglutamine (PAGN), which is excretedin the urine. The structures of PBA, PAA, and PAGN are set forth below.

The clinical benefit of NaPBA and HPN-100 with regard to nitrogenretention disorders derives from the ability of PAGN to effectivelyreplace urea as a vehicle for waste nitrogen excretion and/or to reducethe need for urea synthesis (Brusilow 1991; Brusilow 1993). Because eachglutamine contains two molecules of nitrogen, the body rids itself oftwo waste nitrogen atoms for every molecule of PAGN excreted in theurine. Therefore, two equivalents of nitrogen are removed for each moleof PAA converted to PAGN. PAGN represents the predominant terminalmetabolite, and one that is stoichiometrically related to waste nitrogenremoval, a measure of efficacy in the case of nitrogen retention states.The difference between HPN-100 and NaPBA with respect to metabolism isthat HPN-100 is a triglyceride and requires digestion, presumably bypancreatic lipases, to release PBA (McGuire 2010).

In contrast to NaPBA or HPN-100, sodium benzoate acts when benzoic acidis combined enzymatically with glycine to form hippuric acid. For eachmolecule of hippuric acid excreted in the urine, the body rids itself ofone waste nitrogen atom.

Methods of determining an effective dosage of PAA prodrugs such as NaPBAor HPN-100 for a subject in need of treatment for a nitrogen retentiondisorder are described in WO09/1134460 and WO10/025303. Daily ammonialevels, however, may vary greatly in a subject. This can lead tooverestimation by the physician of the average daily ammonia levels,which may result in overtreatment. Thus, there is a need in the art forimproved methods for PAA prodrug dose determination and adjustment basedon ammonia levels in subjects with nitrogen retention disorders such asUCDs or HE.

SUMMARY

Provided herein in certain embodiments are methods for determiningwhether to increase a dosage of a nitrogen scavenging drug in a subjectwith a nitrogen retention disorder by measuring a fasting blood ammonialevel and comparing the fasting blood ammonia level to the upper limitof normal (ULN) for blood ammonia, where a fasting blood ammonia levelthat is greater than half the ULN for blood ammonia indicates that thedosage needs to be increased. In certain embodiments, the nitrogenretention disorder is a UCD or HE. In certain embodiments, the nitrogenscavenging drug is HPN-100, PBA, NaPBA, sodium benzoate, or anycombination thereof (i.e., any combination of two or more of HPN-100,PBA, NaPBA). In certain embodiments, the ULN is around 35 μmol/L or 59μg/mL. In certain embodiments, the methods include an additional step ofadministering an increased dosage of the nitrogen scavenging drug if theneed exists, and in certain of these embodiments administration of thenitrogen scavenging drug produces a normal average daily ammonia levelin the subject. In certain embodiments wherein a determination is madeto administer an increased dosage of nitrogen scavenging drug andwherein the nitrogen scavenging drug is a PAA prodrug, the methodsinclude an additional step of measuring urinary PAGN excretion anddetermining an effective dosage of the PAA prodrug based on a meanconversion of PAA prodrug to urinary PAGN of 60-75%.

Provided herein in certain embodiments are methods for determiningwhether to administer a nitrogen scavenging drug to a subject with anitrogen retention disorder by measuring a fasting blood ammonia leveland comparing the fasting blood ammonia level to the ULN for bloodammonia, where a fasting blood ammonia level that is greater than halfthe ULN for blood ammonia indicates that the nitrogen scavenging drugneeds to be administered. In certain embodiments, the nitrogen retentiondisorder is a UCD or HE. In certain embodiments, the nitrogen scavengingdrug is HPN-100, PBA, NaPBA, sodium benzoate, or any combination thereof(i.e., any combination of two or more of HPN-100, PBA, NaPBA). Incertain embodiments, the ULN is around 35 μmol/L or 59 μg/mL. In certainembodiments, the methods include an additional step of administering anitrogen scavenging drug if the need exists, and in certain of theseembodiments administration of the nitrogen scavenging drug produces anormal average daily ammonia level in the subject. In certainembodiments wherein a determination is made to administer a nitrogenscavenging drug and wherein the nitrogen scavenging drug is a PAAprodrug, the methods further include a step of determining an effectiveinitial dosage of the PAA prodrug by determining a target urinary PAGNoutput based on a target nitrogen output and calculating an effectiveinitial dosage that results in the target urinary PAGN output based on amean conversion of PAA prodrug to urinary PAGN of 60-75%. In certainembodiments, the methods include a step of administering the calculatedeffective initial dosage.

Provided herein in certain embodiments are methods for treating anitrogen retention disorder in a subject who has previously beenadministered a nitrogen scavenging drug by measuring a fasting bloodammonia level, comparing the fasting blood ammonia level to the ULN forblood ammonia, and administering an increased dosage of the nitrogenscavenging drug if the fasting ammonia level is greater than half theULN for blood ammonia. In certain embodiments, administration of anincreased dosage of the nitrogen scavenging drug produces a normalaverage daily ammonia level in the subject. In certain embodiments, thenitrogen retention disorder is a UCD or HE. In certain embodiments, thenitrogen scavenging drug is HPN-100, PBA, NaPBA, sodium benzoate, or anycombination thereof (i.e., any combination of two or more of HPN-100,PBA, NaPBA). In certain embodiments, the ULN is around 35 μmol/L or 59μg/mL. In certain embodiments wherein the nitrogen scavenging drug is aPAA prodrug, the methods include an additional step of measuring urinaryPAGN excretion and determining an effective dosage of the PAA prodrugbased on a mean conversion of PAA prodrug to urinary PAGN of 60-75%. Incertain embodiments, the methods include a step of administering thecalculated effective dosage.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: The urea cycle and how certain nitrogen-scavenging drugs mayassist in elimination of excessive ammonia.

FIG. 2: Relationship between fasting ammonia and average ammonia UCDpatients.

FIG. 3: Venous blood ammonia values over 24 hours in (A) adult and (B)pediatric UCD patients.

DETAILED DESCRIPTION

The following description of the invention is merely intended toillustrate various embodiments of the invention. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to one skilled in the artthat various equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein.

In subjects with a nitrogen retention disorder, the desired effect oftreatment with a nitrogen scavenging drug is control of blood ammonialevel. Control of blood ammonia level generally refers to ammonia valueswithin the normal range and avoidance of hyperammonemic crises, whichare often defined in the art as transient ammonia values exceeding 100μmol/L or 178 μg/mL accompanied by clinical signs and symptoms ofhyperammonemia. Dosing of nitrogen scavenging drugs is usually basedupon clinical assessment and measurement of ammonia. However, assessmentof treatment effect and interpretation of ammonia levels is confoundedby the fact that individual ammonia values vary several-fold over thecourse of a day and are impacted by timing of the blood draw in relationto the last meal and dose of drug (see, e.g., Lee 2010; Lichter-Konecki2011; Diaz 2011).

A random ammonia value obtained during an outpatient visit may fail toprovide a reliable measure of a subject's status and the drug effect.For example, basing treatment on a blood sample taken after eating ameal might overestimate average daily ammonia level and result inovertreatment. Conversely, basing treatment on a blood sample takenafter drug administration might underestimate average daily ammonialevel and result in undertreatment. A fasting ammonia level at or nearthe ULN might be taken as an indication of satisfactory control withoutappreciating the fact that the ammonia burden during the day (averageand/or highest possible value) might be significantly higher. Thus, afasting level at or near the ULN may actually reflect undertreatment ina subject already a receiving nitrogen scavenging drug or the need fortreatment in a subject not currently prescribed a nitrogen scavengingdrug. A more accurate view of daily ammonia level could be obtained bymultiple blood draws in a controlled setting over an extended period oftime. Although this is currently done in clinical trials, it isclinically impractical.

As set forth below, the relationship between fasting ammonia levels anddaily ammonia exposure was evaluated in subjects with nitrogen retentiondisorders. It was found that fasting ammonia correlates strongly withdaily ammonia exposure, assessed as a 24 hour area under the curve forammonia, daily average, or maximal daily concentration, and that atarget fasting value which does not exceed half of the ULN is aclinically useful and practical predictor of ammonia values over 24hours. As such, provided herein are clinically practical methods ofevaluating ammonia exposure in subjects with nitrogen retentiondisorders based on fasting ammonia levels, as well as methods of usingthe resultant information to adjust the dosage of a nitrogen scavengingdrug, determine whether to administer a nitrogen scavenging drug, treata nitrogen retention disorder, and predict daily ammonia burden. The useof fasting ammonia levels to predict ammonia exposure provides asignificant advantage over previously developed methods by reducing thenumber of required blood draws and eliminating the confusion associatedwith conflicting ammonia levels over the course of the day.

As further disclosed herein, the relationship between ammonia controland neurocognitive outcome was evaluated in UCD patients. Previousresearch has demonstrated that UCD patients often exhibit lower IQoverall and deficient executive function manifested by difficulty ingoal setting, planning, monitoring progress and purposeful problemsolving. As set forth herein, it was found that ammonia control with GPBresulted in a significant improvement in executive functions inpediatric patients. Based on these results, methods are provided hereinfor improving executive function in a pediatric subject with a UCD byadministering one or more nitrogen scavenging drugs.

As further disclosed herein, the relationship between elevated PAAlevels and neurological adverse events (AEs) was analyzed. Many of theover 30 reports of administration of NaPBA and/or sodium PAA to humansdescribe AEs, particularly when administered intravenously. IVadministration of PAA to cancer patients was shown previously to resultin AEs that included fatigue, dizziness, dysgeusia, headache,somnolence, lightheadedness, pedal edema, nausea, vomiting, and rash(Thibault 1994; Thibault 1995). These AEs correlated with PAA levelsfrom 499 to 1285 μg/mL. Although NaPBA has been used in UCD treatmentfor over two decades and AEs reportedly associated with PAA are similarto those associated with hyperammonemia, little was known previouslyabout the relationship between PAA levels and neurological AEs in UCDpatients. As shown herein, increased PAA levels did not correlate withincreased neurological AEs in subjects with UCD. However, PAA levelswere associated with an increase in neurological AEs in healthysubjects. Based on these results, methods are provided herein forpredicting or diagnosing AEs in a subject by measuring PAA levels.Further provided herein are methods of treating and/or preventing AEs ina subject with elevated PAA levels by administering one or more nitrogenscavenging drugs.

Provided herein are specific target values for blood ammonia upon whichan effective dosage of a nitrogen scavenging drug can be based. Incertain embodiments, an effective dosage of a nitrogen scavenging drugmay be an initial dosage, subsequent/maintenance dosage, improveddosage, or a dosage determined in combination with other factors. Incertain embodiments, the effective dosage may be the same as ordifferent than the initial dosage. In other embodiments, the effectivedosage may be higher or lower than the initial dosage. In certainembodiments, methods are provided for adjusting the dose or regimen of anitrogen scavenging drug to achieve a target ammonia level that ispredictive of the average daily ammonia level and/or the highest ammoniavalue that the subject is likely to experience during the day.

Using the methods herein, a subject's fasting blood ammonia level may beused as a predictor of daily ammonia burden, average daily ammonialevel, and/or highest daily ammonia value. Whether a subject with anitrogen retention disorder is receiving an optimum dosage of nitrogenscavenging drug may be determined based on predicted daily ammoniaexposure. By optimizing the therapeutic efficacy of a nitrogenscavenging drug, the therapeutic dosage of the nitrogen scavenging drugis adjusted so that the subject experiences the desired nitrogenscavenging effect. In particular, the dose is adjusted so that thesubject may experience a normal average daily ammonia level. In certainembodiments, the effective dosage of nitrogen scavenging drug isdetermined by adjusting (e.g., increasing) a dosage to achieve a fastingblood ammonia level for a subject that is less than or equal to half theULN for blood ammonia.

Provided herein in certain embodiments are methods of determiningwhether the dosage of a nitrogen scavenging drug needs to be increasedin a subject with a nitrogen retention disorder comprising comparing afasting blood ammonia level for the subject to a ULN for blood ammonia.If the fasting blood ammonia level has a value that greater than halfthe ULN, the dosage of the nitrogen scavenging drug needs to beincreased. In certain embodiments, the methods further compriseincreasing the dosage of the nitrogen scavenging drug if the needexists, and in certain of these embodiments the methods further compriseadministering the increased dosage. In certain of these embodiments,administration of the increased dosage results in a normal average dailyammonia level in the subject.

Provided herein in certain embodiments are methods of determiningwhether the dosage of a nitrogen scavenging drug needs to be increasedin a subject with a nitrogen retention disorder comprising measuring afasting blood ammonia level for the subject and comparing the fastingblood ammonia level to a ULN for blood ammonia. If the fasting bloodammonia level has a value that is greater than half the ULN, the dosageof the nitrogen scavenging drug needs to be increased. In certainembodiments, the methods further comprise increasing the dosage of thenitrogen scavenging drug if the need exists, and in certain of theseembodiments the methods further comprise administering the increaseddosage. In certain of these embodiments, administration of the increaseddosage results in a normal average daily ammonia level in the subject.

Provided herein in certain embodiments are methods of adjusting thedosage of a nitrogen scavenging drug in a subject with a nitrogenretention disorder comprising comparing a fasting blood ammonia levelfor the subject to a ULN for blood ammonia. If the fasting blood ammonialevel has a value that is greater than half the ULN, the dosage of thenitrogen scavenging drug is increased, and if the dosage is less than orequal to half the ULN the dosage of the nitrogen scavenging drug is notincreased. In certain embodiments, the methods further compriseadministering the increased dosage. In certain of these embodiments,administration of the increased dosage results in a normal average dailyammonia level in the subject.

Provided herein in certain embodiments are methods of adjusting thedosage of a nitrogen scavenging drug in a subject with a nitrogenretention disorder comprising measuring a fasting blood ammonia levelfor the subject and comparing the fasting blood ammonia level to a ULNfor blood ammonia. If the fasting blood ammonia level has a value thatis greater than half the ULN, the dosage of the nitrogen scavenging drugis increased, and if the dosage is less than or equal to half the ULNthe dosage of the nitrogen scavenging drug is not increased. In certainembodiments, the methods further comprise administering the increaseddosage. In certain of these embodiments, administration of the increaseddosage results in a normal average daily ammonia level in the subject.

Provided herein in certain embodiments are methods of adjusting thedosage of a nitrogen scavenging drug in a subject with a nitrogenretention disorder comprising measuring a fasting blood ammonia levelfor the subject and comparing the fasting blood ammonia level to a ULNfor blood ammonia. If the fasting blood ammonia level has a value thatis greater than half the ULN, the dosage of the nitrogen scavenging drugis increased, and if the dosage is significantly less than half the ULN,the dosage of the nitrogen scavenging drug may be decreased. In certainembodiments, the methods further comprise administering the adjusteddosage. In certain of these embodiments, administration of the adjusteddosage results in a normal average daily ammonia level in the subject.

Provided herein in certain embodiments are methods of adjusting thedosage of a nitrogen scavenging drug in a subject with a nitrogenretention disorder comprising administering an initial dosage of thenitrogen scavenging drug, measuring fasting blood ammonia level, andcomparing the fasting blood ammonia level to a ULN for blood ammonia. Ifthe fasting blood ammonia level has a value that is greater than halfthe ULN, subsequent maintenance dosages of the nitrogen scavenging drugare adjusted to be greater than the initial dosage. In certainembodiments, the methods further comprise administering the increasedmaintenance dosage, and in certain of these embodiments, administrationof the increased maintenance dosage results in a normal average dailyammonia level in the subject.

Provided herein in certain embodiments are methods of adjusting thedosage of a nitrogen scavenging drug in a subject with a nitrogenretention disorder to achieve a fasting blood ammonia level that is lessthan or equal to half the ULN for blood ammonia comprising measuring afasting blood ammonia level for the subject and comparing the fastingblood ammonia level to a ULN for blood ammonia. If the fasting bloodammonia level has a value that is greater than half the ULN, the subjectis administered an increased dosage of the nitrogen scavenging drug.After a time period sufficient for the drug to reach steady state (e.g.,48 hours, 48 to 72 hours, 72 hours to 1 week, 1 week to 2 weeks, greaterthan 2 weeks), fasting blood ammonia level is measured again andcompared to a ULN for blood ammonia. If the fasting blood ammonia levelhas a value that is greater than half the ULN, the dosage of thenitrogen scavenging drug is increased. This process is repeated until afasting blood ammonia level of less than or equal to half the ULN isobtained.

Provided herein in certain embodiments are methods for assessing whethera subject with a nitrogen retention disorder is more or less likely toneed a dosage adjustment of a nitrogen scavenging drug comprisingmeasuring a fasting blood ammonia level for the subject and comparingthe fasting blood ammonia level to a ULN for blood ammonia, wherein afasting blood ammonia level that is greater than half the value of ULNindicates that the subject is more likely to need a dosage adjustmentand a fasting blood ammonia level less than or equal to half the valueof ULN indicates that the subject is less likely to need a dosageadjustment.

Provided herein in certain embodiments are methods of determiningwhether to administer a nitrogen scavenging drug to a subject withnitrogen retention disorder comprising comparing a fasting blood ammonialevel for the subject to a ULN for blood ammonia. If the fasting bloodammonia level has a value that is greater than half the ULN, a nitrogenscavenging drug needs to be administered to the subject. In certainembodiments, these methods further comprise administering the nitrogenscavenging drug. In certain embodiments, the subject may not have beenadministered any nitrogen scavenging drugs prior to the determination.In other embodiments, the subject may have previously been administereda nitrogen scavenging drug other than the one being evaluated. In theseembodiments, the methods provided herein can be used to determinewhether to administer a new nitrogen scavenging drug to a subject.

Provided herein in certain embodiments are methods of determiningwhether to administer a nitrogen scavenging drug to a subject withnitrogen retention disorder comprising measuring a fasting blood ammonialevel for the subject and comparing the fasting blood ammonia level to aULN for blood ammonia. If the fasting blood ammonia level has a valuethat is greater than half the ULN, a nitrogen scavenging drug needs tobe administered to the subject. In certain embodiments, these methodsfurther comprise administering the nitrogen scavenging drug. In certainembodiments, the subject may not have been administered any nitrogenscavenging drugs prior to the determination. In other embodiments, thesubject may have previously been administered a nitrogen scavenging drugother than the one being evaluated. In these embodiments, the methodsprovided herein can be used to determine whether to administer a newnitrogen scavenging drug to a subject.

Provided herein in certain embodiments are methods for selecting adosage of a nitrogen scavenging drug for treating a nitrogen retentiondisorder in a subject based on blood ammonia levels comprising selectinga dosage that results in a fasting blood ammonia level that is less thanor equal to half the ULN for blood ammonia. In certain embodiments,selecting the effective dosage is further based on diet, endogenouswaste nitrogen excretion capacity, or any combination thereof. Incertain embodiments, the methods further comprise administering theselected dosage.

Provided herein in certain embodiments are methods of treating a subjectwith a nitrogen retention disorder who has previously been administereda nitrogen scavenging drug comprising measuring a fasting blood ammonialevel for the subject and comparing the fasting blood ammonia level to aULN for blood ammonia. If the fasting blood ammonia level has a valuethat is greater than half the ULN, the subject is administered anincreased dosage of the nitrogen scavenging drug. If the fasting bloodammonia level has a value that is less than or equal to half the ULN,the subject is administered the same dosage or a decreased dosage of thenitrogen scavenging drug. In certain embodiments, administration of anincreased dosage results in a normal average daily ammonia level in thesubject.

Provided herein in certain embodiments are methods of treating a subjectwith a nitrogen retention disorder who has previously been administeredan initial dosage of a nitrogen scavenging drug comprising measuring afasting blood ammonia level for the subject and comparing the fastingblood ammonia level to a ULN for blood ammonia. If the fasting bloodammonia level has a value that is greater than half the ULN, the subjectis administered a maintenance dosage that is greater than the initialdosage of the nitrogen scavenging drug. If the fasting blood ammonialevel has a value that is less than or equal to half the ULN, thesubject is administered the initial dosage or a lower dosage. In certainembodiments, administration of an increased maintenance dosage resultsin a normal average daily ammonia level in the subject.

Provided herein in certain embodiments are methods of treating a subjectwith a nitrogen retention disorder comprising administering a nitrogenscavenging drug, then measuring a fasting blood ammonia level for thesubject at some point after drug administration and comparing thefasting blood ammonia level to a ULN for blood ammonia. If the fastingblood ammonia level has a value that is greater than half the ULN, thesubject is administered an increased dosage of the nitrogen scavengingdrug. If the fasting blood ammonia level has a value that is less thanor equal to half the ULN, the subject is administered the original or alower dosage of the drug.

Provided herein in certain embodiments are methods of treating a subjectwith a nitrogen retention disorder comprising administering a firstdosage of a nitrogen scavenging drug, measuring a fasting blood ammonialevel for the subject, and comparing the fasting blood ammonia level toa ULN for blood ammonia. If the fasting blood ammonia level has a valuethat is greater than half the ULN, a second dosage of a nitrogenscavenging drug that is greater than the first dosage is administered tothe subject. A fasting ammonia blood level is measured again in thesubject and compared to a ULN for blood ammonia. If the fasting bloodammonia level has a value that is greater than half the ULN, a thirddosage of a nitrogen scavenging drug that is greater than the seconddosage is administered to the subject. This process is repeated untilthe subject exhibits a fasting blood ammonia level with a value lessthan or equal to half the ULN.

Provided herein in certain embodiments are methods of monitoring theefficacy of nitrogen scavenging drug administration in a subject with anitrogen retention disorder who has previously been administered anitrogen scavenging drug comprising measuring a fasting blood ammonialevel for the subject and comparing the fasting blood ammonia level to aULN for blood ammonia. If the fasting blood ammonia level has a valuethat is greater than half the ULN, the previously administered dosage ofthe nitrogen scavenging drug is considered inadequate to treat thenitrogen retention disorder. If the fasting blood ammonia level has avalue that is less than or equal to half the ULN, the previouslyadministered dosage is considered adequate to treat the nitrogenretention disorder. In certain embodiments where the previouslyadministered dosage is considered inadequate to treat the nitrogenretention disorder, the methods provided herein further compriseadministering an increased dosage of the nitrogen scavenging drug.

Provided herein in certain embodiments are methods for monitoringtherapy with a nitrogen scavenging drug in a subject having a nitrogenretention disorder comprising measuring a fasting blood ammonia levelfrom the subject and comparing the fasting blood ammonia level to a ULNfor blood ammonia, wherein a fasting blood ammonia level that is greaterthan half the ULN indicates that the subject is more likely to need adosage adjustment of the nitrogen scavenging drug, and wherein a fastingblood ammonia level less than or equal to half the ULN indicates thatthe subject is less likely to need a dosage adjustment.

A nitrogen retention disorder as used herein refers to any conditionassociated with elevated blood nitrogen/ammonia levels. In certainembodiments, a nitrogen retention disorder may be a UCD. In otherembodiments, a nitrogen retention disorder may be HE.

A nitrogen scavenging drug as used herein refers to any drug thatdecreases blood nitrogen and/or ammonia levels. In certain embodiments,a nitrogen scavenging drug may remove nitrogen in the form of PAGN, andin certain of these embodiments the nitrogen scavenging drug may be anorally administrable drug that contains or is metabolized to PAA. Forexample, a nitrogen scavenging drug may be a PAA prodrug such as PBA orHPN-100, a pharmaceutically acceptable salt of PBA such as NaPBA, or apharmaceutically acceptable ester, acid, or derivative of a PAA prodrug.In other embodiments, a nitrogen scavenging drug may remove nitrogen viahippuric acid. In certain of these embodiments, a nitrogen scavengingdrug may be benzoic acid, a pharmaceutically acceptable salt of benzoicacid such as sodium benzoate, or a pharmaceutically acceptable ester,acid, or derivative of benzoic acid.

Increasing the dosage of a nitrogen scavenging drug may refer toincreasing the amount of drug per administration (e.g., an increase froma 3 mL dosage to a 6 mL dosage), increasing the number ofadministrations of the drug (e.g., an increase from once-a-day dosing totwice- or three-times-a-day), or any combination thereof.

A subject that has previously been administered a nitrogen scavengingdrug may have been administered the drug for any duration of timesufficient to reach steady state. For example, the subject may have beenadministered the drug over a period of 2 to 7 days, 1 week to 2 weeks, 2weeks to 4 weeks, 4 weeks to 8 weeks, 8 weeks to 16 weeks, or longerthan 16 weeks.

In certain embodiments of the methods disclosed herein, the fastingperiod for obtaining a fasting blood ammonia level is overnight. Incertain embodiments, the fasting period is 4 hours or more, 5 hours ormore, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours ormore, 10 hours or more, 11 hours or more, or 12 hours or more, and incertain embodiments the fasting period is 4-8 hours, 6-8 hours, or 8-12hours. During the fasting period, the subject preferably does not ingestany food. In certain embodiments, the subject may also refrain fromingesting certain non-food substances during the fasting period. Forexample, in certain embodiments the subject does not ingest anysupplements and/or nitrogen scavenging drugs during the fasting period.In certain of these embodiments, the subject may nonetheless ingest oneor more drugs other than nitrogen scavenging drugs during the fastingperiod. In certain embodiments, the subject does not ingest any highcalorie liquids during the fasting period. In certain of theseembodiments, the subject does not ingest any liquids other than waterduring the fasting period. In other embodiments, the subject may ingestsmall amounts of low calorie beverages, such as tea, coffee, or dilutedjuices.

In certain embodiments of the methods disclosed herein, blood samplesused for measuring fasting blood ammonia levels and/or ULN bloodammonias are venous blood samples. In certain embodiments, a bloodsample is a plasma blood sample. Any methods known in the art may beused to obtain a plasma blood sample. For example, blood from a subjectmay be drawn into a tube containing heparin orethylenediaminetetraacetic acid (EDTA). In certain embodiments, thesample can be placed on ice and centrifuged to obtain plasma within 15minutes of collection, stored at 2-8° C. (36-46° F.) and analyzed within3 hours of collection. In other embodiments, the blood plasma sample issnap frozen, stored at <−18° C. (<0° F.) and analyzed at a later time.For example, the sample may be analyzed at 0-12 hours, 12-24 hours,24-48, 48-96 hours after freezing, or within any other timeframe overwhich the sample has demonstrated stability. In certain embodiments,blood samples are taken in a laboratory or hospital setting. In certainembodiments, a single fasting blood sample is used to measure fastingblood ammonia level. However, in other embodiments, multiple fastingblood samples may be obtained. In certain embodiments, a subject's bloodammonia level may be monitored throughout the day. Further, in certainembodiments, the methods disclosed herein comprise an additional step ofobtaining one or more blood samples from a subject prior to or aftermeasuring fasting blood ammonia level.

In certain embodiments, a blood sample is analyzed immediately aftercollection. In other embodiments, the blood sample is stored for someperiod between collection and analysis. In these embodiments, the samplemay be stored for less than 1 hour, 1 hour to 6 hours, 1 hour to 12hours, 1 hour to 24 hours, or 1 hour to 48 hours. In certain of theseembodiments, the blood sample is stored at a temperature between 0-15°C., such as 2-8° C. In other embodiments, the blood sample is storedbelow 0° C. or below −18° C.

Measurement of ammonia levels in a fasting blood sample is carried outusing techniques known in the art. For example, ammonia levels may bemeasured using a colorimetric reaction or an enzymatic reaction. Incertain embodiments, a colorimetric reaction may involve the use ofbromophenol blue as an ammonia indicator. In these embodiments, ammoniamay react with bromophenol blue to yield a blue dye. In certainembodiments, an enzymatic reaction may involve glutamate dehydrogenasecatalyzing the reductive amination of 2-oxoglutarate with NH⁴⁺ and NADPHto form glutamate and NADP⁺. The formation of NADP⁺ formed is directlyproportional to the amount of ammonia present in the blood sample.Therefore, the concentration of ammonia is measured based on a decreasein absorbance.

In certain embodiments of the methods disclosed herein, a subjectexhibiting a fasting blood ammonia level less than or equal to half theULN for blood ammonia has an average likelihood within a confidenceinterval that their average daily ammonia level will remain within anormal average daily ammonia level. In certain embodiments, the averagelikelihood of having a normal daily ammonia value is 80% to 90%. Incertain embodiments, one may predict with 95% confidence that a bloodammonia level will fall within a certain range. In certain embodiments,one can predict with 95% confidence that a true probability ofpredicting normal values based on fasting blood ammonia is between 65%and 93%. In other embodiments, one can predict with 80% confidence thata true probability of predicting normal values based on fasting bloodammonia is at least 70%. In certain embodiments, the average likelihoodof predicting normal ammonia value based on fasting blood ammonia isabout 84% with 95% confidence that the true probability is between 65%and 93%.

In certain embodiments of the methods disclosed herein, a subjectexhibiting a fasting blood ammonia level less than or equal to half theULN for blood ammonia has an average likelihood within a confidenceinterval that their maximum daily blood ammonia level will not exceed1.5 times the ULN for blood ammonia. In certain of these embodiments,the average likelihood is about 70% to 80%. In certain embodiments, theconfidence interval is a 95% confidence interval. In certainembodiments, the average likelihood is about 75% with 95% confidencethat the true probability is between 58% and 86%.

In certain embodiments of the methods disclosed herein, a subjectexhibiting a fasting blood ammonia level less than or equal to half theULN for blood ammonia has an average likelihood within a confidenceinterval that their maximum daily blood ammonia level will be less than100 μmol/L. In certain of these embodiments, the average likelihood is90% to 98%. In certain embodiments, the confidence interval is 95%. Incertain embodiments, the average likelihood is about 93% with 95%confidence that the true probability is between 77% and 100%.

The maximal ammonia value refers to the maximum amount of ammonia thatmay be detected in a subject following consumption of meals, if repeatedmeasurement of blood ammonia can be instituted to detect such maximumvalue over an extended period of time. Based on well-controlled clinicaltrials with repeated blood sampling over 24 hours, the maximum bloodammonia has been observed to occur following the third major meal of theday in the early to mid evening hours (4-8 PM, assuming that breakfastis approximately 8 AM; see, e.g., Lee 2010; Lichter-Konecki 2011).

The ULN for blood ammonia typically represents the highest level in therange of normal values, which may be influenced by a variety of factorssuch as the assay method, types of regents, standard reference samplesused, and specifications and calibration of equipment used to performthe measurement. In certain embodiments of the methods disclosed herein,the ULN for blood ammonia is determined for a subject individually. Inother embodiments, the ULN for blood ammonia may be based onmeasurements obtained across a range of subjects (i.e., subjects withUCD or with a particular subtype of UCD, subjects with HE, healthysubjects, etc.). In certain embodiments, the ULN for blood ammonia mayrepresent a standard reference value disclosed in the art, such as amean ULN developed across a particular subset of subjects. In otherembodiments, the ULN for blood ammonia may represent a standardmeasurement that has been developed by a particular entity that performsblood draws and/or blood evaluations, such as a particular clinicallaboratory. In certain embodiments, the ULN is a standard referencevalue utilized by the same entity that measures the fasting bloodammonia level. In these embodiments, one skilled in the art willappreciate that interpretation of average daily ammonia in subject witha nitrogen retention disorder must be made relative to the referencerange of normal values at the laboratory in which the ammonia wasmeasured. Furthermore, the units of ammonia measurement may also varyfrom lab to lab (e.g., μg/mL or μmoI/L), emphasizing the importance ofinterpreting the subject's ammonia levels relative to the ULN at thelaboratory in which the measurement was performed. In certainembodiments, the ULN for blood ammonia may be in the range of 26-64μmol/L. In certain of these embodiments, the ULN for blood ammonia maybe in the range of 32-38 μmol/L or 34-36 μmol/L, and in certain of theseembodiments the ULN for blood ammonia is 35 μmol/L. In certainembodiments, the ULN for blood ammonia may be in the range of 50-65μg/mL. In certain of these embodiments, the ULN for blood ammonia may bein the range of 55-63 μg/mL or 57-61 μg/mL, and in certain of theseembodiments the ULN for blood ammonia is 59 μg/mL.

In certain embodiments, the average daily ammonia is the average amountof ammonia an individual may experience during the day, if serial bloodsampling were performed for ammonia measurements. In well-controlledclinical studies, it has been established that ammonia fluctuatesseveral fold during the day, depending on the timing of blood drawrelative to food and drug intake. Due to these fluctuations, the timingof individual or serial blood sampling should be controlled relative tothe timing of food and drug intake. Even serial sampling may not beenough to capture the peaks and troughs of the fluctuating ammoniavalues, unless samples are taken frequently enough. Therefore, obtaininga simple average of several measurements may provide inadequate ormisleading information regarding the total ammonia burden a subject mayexperience during the day.

Provided herein are methods to better estimate a subject's average dailyammonia assessed as the area under the curve for 24-hr ammonia (ammoniaAUC_(0-24hr)) obtained from adequate and well-spaced samples over 24hours. This ammonia AUC_(0-24hr) can be further normalized for theentire actual period of sampling, i.e., ammonia AUC_(0-24hr) is dividedby the sampling period (e.g., 24 hours). For example, if an AUC of 1440μmol*hr/L is calculated using the trapezoidal rule based on 8-11 ammoniavalues obtained over 24 hours, then the average daily ammonia value ortime-normalized AUC_(0-24hr) would be equal to 1440 μmol*hr/ml dividedby the sampling time of 24 hr, or 60 μmol/L. If the normal referencerange at the laboratory which performed the ammonia analysis was 10-35μmol/L, then the average daily ammonia value for this subject would beapproximately 1.71 times the ULN of 35 μmol/L. Similarly, if the ammoniaAUC_(0-24hr) was determined to be equal to 840 μmol*hr/L based onmultiple, well-spaced samples over 24 hours and analyzed at the samelaboratory, and the sampling period was 24 hours, then thetime-normalized AUC_(0-24hr) would be 35 μmol/L. This corresponds to anaverage ammonia or daily ammonia burden within the ULN. Finally,subjects with nitrogen retention disorders such as UCDs may experience ahyperammonemic crisis, which is often defined clinically as a bloodlevel exceeding 100 μmol/L and clinical manifestations ofhyperammonemia, which may require intervention to prevent irreversiblehard and enable recovery.

Provided herein are methods of adjusting nitrogen scavenging drug dosageby measuring fasting blood ammonia to minimize the likelihood a subjectmay experience an ammonia value (Cmax) over 24 hours that exceeds 100μmol/L. It has been found that 100 μmol/L corresponds to approximately2-3 times the ULN in most laboratories. Previously, if a subject with anitrogen retention disorder such as UCD had a blood ammonia level withinor slightly above the normal reference range for the laboratory whichperformed the analysis, the subject was considered to be in goodclinical control regardless of the timing of the blood draw in relationto meals and last administration of drug dose. However, it has beenshown that a subject with a UCD who has a fasting blood ammonia levelbetween the ULN and 1.5 times the ULN (e.g., 35 to 52 μmol/L) has anaverage likelihood of only 45% (with a 95% confidence interval of 21% to70%) that his or her average daily ammonia is within the normal range;an average likelihood of only 35% (with a 95% confidence interval of 13%to 60%) that his or her maximal level of ammonia during the day is lessthan 1.5 times the ULN (e.g., 52 μmol/L); and an average likelihood of25% that his or her maximal daily ammonia level exceeds 100 μmol/Lduring the day. Thus, after measuring a UCD subject's fasting bloodammonia, the dosage of a nitrogen scavenging drug may be progressivelyincreased and/or his or her protein intake progressively decreased untilthe fasting ammonia value is less than or equal to half of the ULN forthe local laboratory in which the ammonia analysis was performed.

In certain embodiments of the methods disclosed herein, one or morefactors other than ammonia level may be taken into consideration whenevaluating nitrogen scavenging drug dosage. For example, blood ammoniameasurements may be combined with urinary PAGN measurements indetermining whether to administer a nitrogen scavenging drug, adjustingthe dosage of a nitrogen scavenging drug, or treating a nitrogenretention disorder. US Patent Publication No. 2010/0008859 disclosesthat urinary PAGN levels correlate more closely to PBA prodrug dosagethan plasma PAA, PBA, or PAGN levels, and further discloses that PBAprodrugs are converted to urinary PAGN with a mean efficiency of 60-75%.Therefore, certain embodiments of the methods disclosed herein comprisean additional step wherein urinary PAGN levels are measured. In certainof these embodiments, calculation of an effective dosage of nitrogenscavenging drug is based in part on a mean 60-75% conversion of PAAprodrug to urinary PAGN. For example, in certain embodiments the methodsdisclosed herein for determining whether to administer a nitrogenscavenging drug to a subject comprise an additional step of measuringurinary PAGN and calculating an effective initial dosage based on a meanconversion of PAA prodrug to urinary PAGN of 60-75%. Similarly, incertain embodiments the methods disclosed herein for adjusting thedosage of a nitrogen scavenging drug comprise an additional step ofmeasuring urinary PAGN and calculating an effective dosage based on amean conversion of PAA prodrug to urinary PAGN of 60-75%. In certain ofthese embodiments, the effective dosage is calculated based on a targetnitrogen output. In certain embodiments, urinary PAGN may be determinedas a ratio of the concentration of urinary PAGN to urinary creatinine.In certain embodiments, urinary PAGN is a factor that is taken intoconsideration when determining whether to administer or increase thedosage of a nitrogen scavenging drug, i.e., urinary PAGN is evaluated incombination with ammonia level to determine whether to administer orincrease the dosage of the drug. In other embodiments, ammonia levelalone is used to determine whether to administer or increase the dosageof a nitrogen scavenging drug, and urinary PAGN is simply used tocalculate the initial or adjusted dosage.

One skilled in the art will recognize that a variety of other factorsmay be taken into consideration when determining the effective dosage ofa nitrogen scavenging drug. For example, factors such as diet (e.g.,protein intake) and endogenous waste nitrogen capacity (e.g., ureasynthesis capacity) may be considered.

Provided herein in certain embodiments are kits for carrying out themethods disclosed herein. In certain embodiments, kits are provided fordetermining whether to administer or adjust the dosage of a nitrogenscavenging drug for a subject with a nitrogen retention disorder. Thekits disclosed herein may include one or more nitrogen scavenging drugsand/or one or more reagents (e.g., bromophenol blue) or enzymes (e.g.,glutamate dehydrogenase) to measure blood ammonia levels in a sample.The kit may additionally include other pigments, binders, surfactants,buffers, stabilizers, and/or chemicals necessary to obtain a bloodsample and to measure the ammonia level in the sample. In certainembodiments, the kits provided herein comprise instructions in atangible medium.

One of ordinary skill in the art will recognize that the variousembodiments described herein can be combined.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention. It will be understood thatmany variations can be made in the procedures herein described whilestill remaining within the bounds of the present invention. It is theintention of the inventors that such variations are included within thescope of the invention.

EXAMPLES Example 1: Analysis of Predictability of PharmacodynamicAmmonia Values from Fasting Ammonia in UCD Patients

This example demonstrates the relationship between fasting ammonia andthe pharmacodynamic (PD) profile of daily ammonia in patients receivingPAA prodrugs for UCDs. Ammonia values vary many-fold over the course of24 hours in UCD patients. As depicted in FIGS. 3a and 3b , venousammonia was measured for 24 hours following one week of dosing witheither NaPBA or glycerol phenylbutyrate (GPB). The graphs displayammonia values as mean±SD over 24 hours, where time zero corresponds tojust prior to dosing and breakfast (i.e., fasting state). In view ofthis variability in daily ammonia levels, a single measurement may notbe very informative in determining whether a UCD patient is optimallydosed. The ability to predict the highest potential ammonia a UCDpatient may experience during the day and the average 24-hour ammoniafrom a single measurement such as fasting levels has important practicalimplications for nitrogen scavenging drug dosing guidelines and patientmanagement.

Data from two Phase 2 studies and one Phase 3 study comparing ammoniacontrol assessed by 24-hour sampling during steady state treatment withHPN-100 versus NaPBA in 65 UCD patients were used for the analysis. Thetwo Phase 2 studies include protocols UP 1204-003 and HPN-100-005 (Lee2010; Lichter-Konecki 2011). The Phase 3 study includes protocols fromHPN-100-006 (Diaz 2011).

Ammonia values obtained from different hospital laboratories withdifferent normal ranges were normalized to a standard laboratory rangeof 9-35 μmol/L. The patient population included a broad range of ages,UCD subtypes, and doses of drug, and is summarized in Table 1 below.

TABLE 1 UCD demographics in studies UP 1204-003, HPN-100-005, andHPN-100-006: Gender Male 18 (27.7) n (%) Female 47 (72.3) Age atscreening N 65 (years) Mean (SD) 29.46 (15.764) Median   24.00 Range6.0-75.0 UCD diagnosis OTC deficiency 57 (87.7) n (%) CPS1 deficiency 1(1.5) ASS deficiency 5 (7.7) ASL deficiency 1 (1.5) Missing 1 (1.5)Duration of NaPBA N 63 treatment Mean (SD) 114.14 (90.147)   (months)Median 101.00 Range  0.2-300.0 Daily dose NaPBA N 64 Mean (SD) 14.10(6.255)   Median   13.50 Range 1.5-36.0

Exploratory Analysis:

Several PD parameters for steady-state ammonia were explored:AUC_(0-24hr), time-normalized AUC, log AUC, maximal ammonia value over24 hours (Cmax), and average ammonia. Data from 65 subjects from allthree studies with steady-state ammonia and fasting ammonia were used.Missing data were imputed per procedures specified in the protocol andstatistical analysis plan, except that no imputations were made forsubjects who had no PK sampling conducted while on a given study drug.

Sample collection times of 0-hr (before first daily dose) and 24-hourspost-dose (before first daily dose of the following day) were bothevaluated as representative of fasting ammonia. No noticeable differencein the shape or quality of the relationship due to the choice of timepoint was observed.

The relationship between fasting ammonia and pharmacokinetic profile wasevaluated separately for HPN-100 and NaPBA, with no apparent differencein the strength or magnitude of the relationship. Therefore, all datafrom both HPN-100 and NaPBA treatments were used and conclusionsregarding fasting ammonia pertain to both HPN-100 and NaPBA.

The relationships between (1) fasting ammonia and AUC_(0-24hr) and (2)fasting ammonia and maximum observed ammonia (Cmax) were visuallyexplored for the whole population. The effects of the followingcovariates were also observed: age, weight, gender, and dietary proteinintake. A positive and strong relationship was observed between fastingammonia and AUC_(0-24hr), with increasing fasting ammonia beingassociated with higher AUC_(0-24hr) and maximum observed ammonia (FIG.2).

Prediction of AUC_(0-24hr) Through GEE Modeling:

The aim of this modeling was to predict average daily or highestachieved ammonia based on the subject's fasting ammonia. In order totake into account the differences in normal ranges at differentlaboratories, all ammonia values were normalized to a reference range of9-35 μmol/L, and the predictions were referenced to the ULN rather thana fixed value.

Generalized Estimating Equations (GEE) were used to model the predictiveability of fasting ammonia against various ammonia PD properties. GEEmethodology can be used to analyze repeated measures of categoricaldata, in which the repeated measures are assumed to be correlated (Liang1986). The model allows for the specification of the assumed correlationstructure without the knowledge of the magnitude of the correlation.

The 24-hour ammonia profile was divided into ordered categories using avariety of endpoints and cutpoints as follows:

-   -   1) AUC [0-1.0*ULN, >1.0*ULN];    -   2) AUC [0-1.5*ULN, >1.5*ULN];    -   3) Cmax [0-1.0*ULN, >1.0*ULN];    -   4) Cmax [0-1.5*ULN, >1.5*ULN]; and    -   5) Cmax [0-100] μmol/L.

Three levels of fasting ammonia were considered in separate models asinput:

-   -   1) [0-0.5*ULN];    -   2) [>0.5*ULN-<1.0 ULN]; and    -   3) [>1.0*ULN-1.5*ULN].

Using Statistical Analysis Software (SAS) Proc Genmod, generalizedlinear models were fit with a logit link function. Pre-dose fastingammonia was the only predictor variable in the model. The repeatednature of the data (two study periods per subject) was modeled using GEEwith exchangeable correlation matrix. ULN for fasting ammonia was set at35 μmol/L. ULN for AUC over 24 hours was taken as 840 (35 μmol/L*24hours); i.e., the AUC which corresponds to an average daily ammonia lessthan or equal to 35 μmol/L, which was the normalized ULN among theparticipating study sites and is derived by dividing the 24-hour areaunder the curve by the sampling time of 24 hours. The GEE model wasbootstrap-resampled 1,000 times according to the method outlined inDavison, A. C. & Hinkley, D. V., Bootstrap Methods and theirApplication, Cambridge University Press, London (1997), pp. 358-362. Theresults of these models are shown in Table 2 below.

TABLE 2 Summary of results from GEE model to predict ability of fastingammonia against various ammonia PD properties: Bootstrap FastingProbability of pred. error ammonia Ammonia outcome in BootstrapBootstrap rate* Model # level PK outcome category 95% c.i. 80% c.i. (%)1 [0-0.5 AUC in 24 0.84 0.67, 0.93 0.71, 0.89 11.5 ULN] hours [0-1.0ULN] 2 AUC in 24 Did not converge hours [0-1.5 ULN] 3 Cmax 0.53 0.38,0.65 0.42, 0.61 45.8 observed [0-1.0 ULN] 4 Cmax 0.76 0.61, 0.86 0.66,0.82 23.3 observed [0-1.5 ULN] 5 Cmax 0.93 0.78, 1.00 0.85, 0.97 5.7observed [0-100] 6 [0-<1.0 AUC in 24 0.58 0.42, 0.73 0.48, 0.68 42.8ULN] hours [0-1.0 ULN] 7 AUC in 24 0.88 0.78, 0.97 0.82, 0.94 11.1 hours[0-1.5 ULN] 8 AUC in 24 0.97 0.90, 1.00 0.93, 1.00 2.2 hours [0-2 ULN] 9Cmax 0.21 0.11, 0.38 0.14, 0.33 20.0 observed [0-1.0 ULN] 10 Cmax 0.520.35, 0.66 0.42, 0.61 46.0 observed [0-1.5 ULN] 11 Cmax 0.74 0.62, 0.850.91, 1.00 27.2 observed [0-2.0 ULN] 12 Cmax 0.95 0.88, 1.00 0.66, 0.814.3 observed [0-100] 13 [>1.0-1.5 AUC in 24 0.45 0.24, 0.71 0.30, 0.6343 ULN] hours [0-1.0 ULN] 14 AUC in 24 Did not converge hours [0-1.5ULN] 15 AUC in 24 0.80 0.49, 0.99 0.63, 0.92 27 hours [0-2 ULN] 16 CmaxDid not converge observed [0-1.0 ULN] 17 Cmax 0.35 0.16, 0.58 0.23, 0.5133 observed [0-1.5 ULN] 18 Cmax Did not converge observed [0-2.0 ULN] 19Cmax Did not converge observed [0-100]

From Table 2 above, we can conclude that in the population of UCDpatients described in Table 1, we can be 95% confident that, given afasting ammonia less than or equal to half the ULN, the true probabilityof having an AUC in the range [0-840] is on average 84%, at least 67%,and as high as 93%.

Row 1 of Table 2 above suggests that a UCD patient with a fastingammonia of 17 μmol/L as determined by a laboratory with a normalreference range of 9-35 μmol/L (i.e., a fasting ammonia in the range[0-0.5 ULN]) has an 84% chance (with a 95% confidence interval of 67% to93%) of having a time normalized AUC_(0-24hr) in the normal range[AUC_(0-24hr) of 0-840 or an average daily ammonia of 35 μmol/L], a 76%chance (with a 95% confidence interval of 61% to 86%) of having a Cmaxof less than 1.5 ULN, and a 93% chance (with a 95% confidence intervalof 78% to 100%) of never having an ammonia of more than 100 μmol/L.Therefore, this patient would be optimally controlled and unlikely tosuffer from high ammonia during the day.

This Example shows that fasting ammonia correlates strongly with dailyammonia exposure, assessed as a daily average or as maximal dailyconcentration, and that a target fasting value which does not exceedhalf of the upper level of normal for the local lab appears to be aclinically useful as well as practical predictor of ammonia values over24 hours as well. Furthermore, this Example shows that a subject with afasting ammonia in the range 0-0.5 ULN has an 84% chance of having anAUC_(0-24hr) in the normal range (0-840 or an average daily ammonia of35 μmol/L).

Example 2: Selecting and Adjusting HPN-100 Dosage Based on Fasting BloodAmmonia Levels in a Patient with UCD

Patient A is an adult with UCD being managed with amino acid supplementsand dietary protein restriction only. Patient A consumes neither hissupplements nor food for approximately 8 hours prior to a fastingmorning blood draw. A venous blood draw is performed, and fasting bloodammonia level is determined to be 52 μmol/L. This fasting blood ammonialevel is compared to the ULN for blood ammonia in the laboratoryperforming the blood draw, which is 35 μmol/L. Based on the correlationof fasting ammonia level to average ammonia level, it is determined thatPatient A's fasting blood ammonia level of approximately 1.5 times theULN represents only a 45% chance on average of having an average ammoniaduring the day within the normal range. Thus, the ratio of fasting bloodammonia level to ULN for blood ammonia indicates that Patient A willbenefit from treatment with a nitrogen scavenging drug.

The physician elects to treat Patient A with HPN-100. Initial dosage isdetermined based on body surface area or as otherwise instructedaccording to HPN-100 drug labeling. Patient A's body surface area is 1.4m², and therefore the initial dosage is determined to be 9 mL per day or3 mL TID, which is approximately 60% of the maximum allowed dosage perHPN-100 label. Patient A is treated with 9 mL/day of HPN-100 for atleast 7 days, and returns for an additional blood draw. The fastingblood ammonia level at this time is 33 μmol/L, which is slightly belowthe ULN and falls into the range of 0.5 to 1.0 times normal. Patient A'sblood ammonia level is monitored throughout the day after administrationof a 3 mL dose of HPN-100 with each meal. It is observed that PatientA's maximum ammonia reaches 95 μmol/L after dinner with an average dailyammonia of 66 μmol/L, which is almost two times the upper normal range.Therefore, Patient A's dosage of HPN-100 is increased by approximatelyone-third to 12 mL total or 4 mL TID. Patient A returns after at least 7days of treatment with HPN-100. Patient A's fasting ammonia level is 15μmol/L, which is less than half of the ULN range. It is determined thatPatient A has reached satisfactory ammonia control.

It is expected that if Patient A adheres to his prescribed diet, hismaximal daily ammonia is not expected to exceed approximately 52 μmol/L,i.e., approximately 1.5 times the ULN, with an average likelihood of 75%with 95% confidence. The average ammonia level during the day isexpected to remain within normal range with greater than 84% likelihoodand 95% confidence. Moreover, Patient A's maximal daily ammonia ishighly unlikely to reach 100 μmol/L during the day.

Example 3: Adjusting HPN-100 Dosage Based on Fasting Blood AmmoniaLevels in a Patient with UCD

Patient B is an 11-year UCD patient receiving 24 pills of BUPHENYL® perday, amino acid supplements, and restricted dietary protein intake.Patient B does not consume BUPHENYL®, supplements, or food forapproximately 6 hours prior to a fasting morning blood draw. A venousblood draw is performed, and fasting blood ammonia level is determinedto be 40 μmol/L. This fasting blood ammonia level is compared to the ULNfor blood ammonia for the laboratory performing the blood draw, which is35 μmol/L. Based on the correlation of fasting ammonia level to averageammonia level, it is determined that Patient B's fasting blood ammonialevel falling between 1 and 1.5 times the ULN represents a 55% chance ofhaving an average ammonia during the day that is greater than the normalrange, and as high as a 65% chance that her ammonia will go above 52μmol/L or 1.5 times ULN during the day.

Based on discussion with the patient and her mother, the physiciansuspects that Patient B is noncompliant with her medication, and decidesto change her to HPN-100. The initial dosage is determined based on theamount of BUPHENYL® Patient B was receiving, and it is determined thatPatient B needs to take 10.5 mL of HPN-100 per day. Patient B is treatedwith 3.5 mL of HPN-100 3 times a day for at least 7 days, and returnsfor additional blood draws. Her fasting blood ammonia level at this timeis 17 μmol/L, which is below the ULN and falls into the range of 0 to0.5 times normal. It is determined that Patient B has reachedsatisfactory ammonia control.

It is expected that if Patient B adheres to her prescribed diet, hermaximal daily ammonia will not go above approximately 50 μmol/L, whichis less than 1.5 times the ULN. Her average ammonia level during the dayis expected with greater than 84% average likelihood to remain withinnormal range. Moreover, there is only a small chance (7%) that PatientB's maximal daily ammonia will exceed 100 μmol/L during the day.

Example 4: Selecting and Adjusting Sodium Benzoate Dosage Based onFasting Blood Ammonia Levels in a Patient with UCD

Patient C is an adult UCD patient who is allergic to PBA and istherefore being managed with amino acid supplements and dietary proteinrestriction only. Patient C complains of chronic headache and frequentnausea. Patient C consumes neither his supplements nor food forapproximately 8 hours prior to a fasting morning blood draw. A venousblood draw is performed, and fasting blood ammonia level is determinedto be 77 μmol/L. This fasting blood ammonia level is compared to the ULNfor blood ammonia for the laboratory performing the blood draw, which is35 μmol/L. Based on the correlation of fasting ammonia level to averageammonia level, it is determined that Patient C's fasting blood ammonialevel of approximately 2 times the ULN represents a high likelihood ofammonia levels going over 100 μmol/L during the day. Thus, the ratio offasting blood ammonia level to ULN for blood ammonia indicates thatPatient C will benefit from treatment with a nitrogen scavenging drug.

The physician decides to treat Patient C with 15 g of sodium benzoateper day since the patient is allergic to PBA. Patient C is treated with15 g/day of sodium benzoate for at least 7 days, and returns foradditional blood draws. Fasting blood ammonia level at this time is 35μmol/L, which is equal to the ULN. Patient C's dosage of sodium benzoateis increased by approximately 30% to 18 grams per day. After at least 7days of treatment, Patient C's fasting ammonia level is 15 μmol/L, whichis less than half of the ULN. It is determined that Patient C hasreached satisfactory ammonia control.

It is expected that if Patient C adheres to his prescribed diet andmedication, his maximal daily ammonia will not exceed approximately 52μmol/L, which is approximately 1.5 times the ULN. His average ammonialevel during the day is expected with greater than 80% likelihood toremain within normal range. Moreover, Patient C's maximal daily ammoniais highly unlikely to reach 100 μmol/L during the day.

Example 5: Evaluation of the Effect of Ammonia Control on NeurocognitiveOutcome

It has been shown that UCD patients are likely to suffer from diminishedintelligence and impaired neurocognitive functions (Kirvitsky 2009).These neuropsychological impairments have been attributed to repeatedepisodes of acute hyperammonemia interspersed on chronically elevatedammonia. Abnormalities in neuropsychological function and/or brainimaging have been detected even in UCD patients with mild disorders whoexhibit normal IQ and/or appear clinical normal (Gropman 2008a; Gropman2008b). Therefore, it was hypothesized that maintaining average dailyammonia within normal limits and thereby reducing the long term ammoniaburden could result in improved cognition.

The relationship between reducing ammonia burden by maintaining fastingammonia at or close to half ULN and neuropsychological outcomes inpediatric UCD patients was explored in clinical trials. Eleven pediatricpatients ages 6-17 were enrolled in short term switch over comparison ofNaPBA and HPN-100 in controlling ammonia. These patients underwent 24-hrserial sample collection in a confined setting where the last sample at24 hr was considered fasting and under supervision of the studypersonnel. At the end of treatment with HPN-100 the average fastingammonia at 24-hr time point was 15.5 μmol/L or less than half ULN,indicating good clinical control. These 11 patients along with another15 pediatric patients were enrolled in two long term studies andreceived HPN-100 for 12 months, during which monthly fasting ammoniawere collected. At the time of enrollment and at the end of the study,all patients underwent assessment for neuropsychological outcomesincluding the following: BRIEF (Behavior Rating Inventory of ExecutiveFunction) to assess day-to-day executive functioning, CBCL (ChildBehavior Checklist) to evaluate internalizing (e.g., mood/anxiety) andexternalizing behaviors, and WASI (Wechsler Abbreviated Scale ofIntelligence) to estimate of intellectual ability.

During the 12 month treatment with HPN-100, pediatric UCD patientsexperienced fewer episodes of acute hyperammonemia than in the 12 monthspreceding enrollment (5 episodes during the study versus 9 beforeenrollment), with peak ammonia dropping from a mean of 233 μmol/L beforeenrollment to 166 μmol/L during the study. Fasting ammonia remainedcontrolled and monthly averages were at or close to half ULN, rangingfrom 17 to 22 μmol/L. Although patients had been instructed to remainfasting before monthly study visits, some ammonia samples were taken ina non-fasted state, resulting in average monthly ammonia of slightlyabove half ULN.

In pediatric patients, WASI and CBCL scores were stable in comparison tobaseline. The majority of the BRIEF subscales at baseline were at orclose to 65, consistent with borderline and/or clinically significantdysfunction. Among 22 pediatric subjects who completed theneuropsychological testing at 12 months, all BRIEF domains were improved(lower T scores) with means (SD) at end of study compared to baselinefor Behavioral Regulation Index 53.7 (9.79) vs. 60.4 (14.03) (p<0.05);Metacognition Index 57.5 (9.84) vs. 67.5 (13.72) (p<0.001), and GlobalExecutive Scale 56.5 (9.71) vs. 66.2 (14.02) (p<0.001).

The significant improvement in executive functions in this group ofpediatric UCD patients indicates the importance of long term ammoniacontrol and achieving target levels of fasting ammonia.

Example 6: Correlation of Elevated PAA Levels to Neurological AEs in UCDand Healthy Subjects

Elevated plasma levels of PAA may cause symptoms that mimic thoseassociated with hyperammonemia, including headache, nausea, somnolence,etc. Since such symptoms are common and nonspecific, an ammonia levelbelow half the upper limit of normal in a subject with a nitrogenretention disorder who exhibits such symptoms and is receiving a PAAprodrug would prompt a physician to check plasma PAA levels.

The relationship between elevated PAA levels and neurological AEs wasevaluated in three populations: (1) 130 healthy adults dosed with 4 to12 mL TID of GPB in a thorough QTc study, (2) 54 adult and 11 pediatricUCD patients (ages 6-17) enrolled in one of 3 protocols involving shortterm (2-4 week) switchover comparisons of NaPBA vs. GPB, and (3) 77patients enrolled in two nearly identical 12-month GPB treatmentprotocols. In populations 1 and 2, maximal PAA (i.e., Cmax) levels wereanalyzed in relation to neurological AEs as defined by MEDDRA using anExact non-parametric Mann-Whitney test and Generalized EstimatingEquations (GEE) with a logit link function and effects for dose and PAAlevel. The relationship between PAA levels and the occurrence of the AEsreported by Thiebault was also explored in population 3.

No statistically significant relationship was observed betweenneurological AEs and PAA levels for either GPB or NaPBA. The odds ratioof a neurological AE occurring for each 20 μg/mL increase in PAA levelsfor the two drugs combined was 0.95, very close to 1. Thus, among UCDpatients dosed with HPN-100 or NaPBA over the ranges used in thesestudies, increasing levels of PAA (ranging up to 244 μg/mL) were notassociated with an increase in neurological AEs. Similarly, inpopulation 3, PAA levels did not increase over time and exhibited noapparent relationship to neurological AEs, which also did not increasein frequency over time. The pediatric patient with the highest PAA level(410 μg/mL) did not report neurological AEs close to the timing of theblood draw.

Unlike UCD subjects, healthy adult volunteers who reported a nervoussystem AE had statistically significantly higher PAA C_(max) levels thanthose who did not. While this analysis in healthy adults is compromisedby the fact that PAA levels were not always available at the time ofoccurrence of the AEs, as well as by the small sample size in the higherdose groups, the odds ratio of 1.75 (p=0.006) suggests that increasinglevels of PAA are associated with increased probability of experiencinga nervous system AE among healthy adults. AEs reported by healthy adultsgenerally began within 36 hours of dosing and, among those adults whoremained on study, most resolved with continued dosing.

A significant relationship between PAA levels and occurrence ofneurological AEs, which generally resolved with continued dosing, wasdetected in healthy volunteers. Unlike in healthy adults, PAA C_(max)did not correlate with nervous system AEs in UCD patients over a similarrange of doses and PAA levels. These findings may reflect metabolicdifferences among the populations (e.g., UCD patients exhibit highglutamine levels compared with healthy humans) and/or metabolicadaptation with continued dosing.

Population PK model building was performed on 65 UCD patients whoparticipated in the short-term switchover Hyperion studies using NONMEM(version 7.2) based on 2981 ([PBA], [PAA], [PAGN], and urine PAGN[UPAGN])) data points from 53 adult and 11 pediatric UCD patients (ages6-17) who participated in 3 switchover studies of NaPBA and GPB. Themedian GPB dose, expressed as grams of PBA per m2, was 8.85 and 7.01 forpediatric and adult subjects, respectively. Diagnostic plots andstatistical comparisons were used to select among candidate models, andcovariates were assessed by graphical analyses and covariate modeling.Using the final popPK model and parameter estimates, Monte Carlosimulations were performed in ˜1000 virtual patients for a range ofNaPBA and GPB doses to predict systemic metabolite exposure and UPAGNoutput.

The final model that best fit the data was characterized by (a) partialconversion of PBA to PAGN prior to reaching the systemic circulation,(b) saturable conversion of PAA to PAGN (Km ˜161 ug/ml), and (c) ˜60%slower PBA absorption when delivered as GPB vs. NaPBA. Body surface area(BSA) was a significant covariate such that metabolite clearance wasproportionally related to BSA. Fractional presystemic metabolism of PBAwas higher for adults than for pediatric patients receiving GPB (43% vs.14%), whereas the reverse was true for NaPBA (23% vs. 43%). Predictedmedian PAA exposure based on simulated GPB dosing at the PBA equivalentof 13 g/m2 of NaPBA was ˜13%-22% lower in adults than NaPBA (Cmax=82 vs.106 μg/mL; AUC₀₋₂₄=649 vs. 829 μg·h/m) and ˜13% higher in pediatricsubjects ages 6-17 than NaPBA (Cmax=154 vs. 138 μg/mL; AUC₀₋₂₄=1286 vs.1154 μg·h/ml); predicted upper 95th percentile PAA exposure was below500 μg/mL and 25%-40% lower for adult subjects on GPB versus NaPBA andsimilar for pediatric subjects. Simulated dosing at the PBA equivalentof ˜5 g/m² of NaPBA yielded similar and less variable PAA exposure forboth drugs and for pediatric and adult patients. Recovery of PBA asUPAGN was very similar whether delivered orally as GPB or NaPBA.

These findings based on PopPK modeling and dosing simulations suggestthat while most patients treated with PAA prodrugs including NaPBA orHPN-100 will have PAA levels below those reportedly associated withtoxicity and while no relationship between PAA levels and neurologicalAEs was found on a population basis, individual patients exhibitingsymptoms such as headache or nausea might be suffering from eitherhyperammonemia or high PAA levels and that a fasting ammonia level equalto or below half the upper limit of normal would prompt the physician tocheck plasma PAA levels.

As stated above, the foregoing is merely intended to illustrate variousembodiments of the present invention. The specific modificationsdiscussed above are not to be construed as limitations on the scope ofthe invention. It will be apparent to one skilled in the art thatvarious equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein. All referencescited herein are incorporated by reference as if fully set forth herein.

REFERENCES

-   1. Brusilow Science 207:659 (1980)-   2. Brusilow Pediatr Res 29:147 (1991)-   3. Diaz Mol Genet Metab 102:276 (2011)-   4. Gropman Mol Genet Metab 94:52 (2008a)-   5. Gropman Mol Genet Metab 95:21 (2008b)-   6. Lee Mol Genet Metab 100:221 (2010)-   7. Liang Biometrika 73:13 (1986)-   8. Lichter-Konecki Mol Genet Metab 103:323 (2011)-   9. McGuire Hepatology 51:2077 (2010)-   10. Thibault Cancer Res 54:1690 (1994)-   11. Thibault Cancer 75:2932 (1995)

1-11. (canceled)
 12. A method of treating a subject with a urea cycledisorder (UCD) who has a fasting plasma ammonia level less than theupper limit of normal and who is not experiencing a hyperammonemiccrisis, the method comprising: a) orally administering an initial dosageof glyceryl tri[4-phenylbutyrate]; b) after a time period sufficient forthe glyceryl tri-[4-phenylbutyrate] to reach steady state, measuring asingle fasting plasma ammonia level for the subject and not serial blooddraws; c) comparing the fasting plasma ammonia level to the upper limitof normal, wherein the upper limit of normal is relative to thereference range of normal values at the laboratory in which the ammoniawas measured; and d) orally administering an adjusted dosage of glyceryltri-[4-phenylbutyrate], wherein the adjusted dosage is greater than theinitial dosage if the fasting plasma ammonia level is greater than halfthe upper limit of normal for plasma ammonia level, wherein a subjectwith a fasting ammonia in the range 0-0.5 ULN has a greater than 80%likelihood of having an average daily ammonia level within a normalrange, wherein said method further comprises informing the subject thatadministration of the glyceryl tri-[4-phenylbutyrate] can result inelevated phenylacetic acid (PAA) levels and/or neurological adverseevents.
 13. The method of claim 12, wherein the time period sufficientfor the glyceryl tri-[4-phenylbutyrate] to reach steady state is 48hours.
 14. The method of claim 12, wherein the time period sufficientfor the glyceryl tri-[4-phenylbutyrate] to reach steady state is 48 to72 hours.
 15. The method of claim 12, wherein the time period sufficientfor the glyceryl tri-[4-phenylbutyrate] to reach steady state is 72hours to 1 week.
 16. The method of claim 12, wherein the time periodsufficient for the glyceryl tri-[4-phenylbutyrate] to reach steady stateis 1 week to 2 weeks.
 17. The method of claim 12, wherein the timeperiod sufficient for the glyceryl tri-[4-phenylbutyrate] to reachsteady state is greater than 2 weeks.
 18. The method of claim 12,further comprising repeating steps (b) to (d) until the subject exhibitsa fasting plasma ammonia level at or below half the upper limit ofnormal for plasma ammonia level.
 19. The method of claim 12, wherein theupper limit of normal for plasma ammonia level is 35 μmol/L.
 20. Themethod of claim 12, wherein the fasting period for obtaining a fastingblood ammonia level is overnight.
 21. The method of claim 12, furthercomprising monitoring the subject for elevated PAA levels and/orneurological adverse events.
 22. The method of claim 12, furthercomprising measuring the subject's PAA level.